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Differential effect of hyperglycaemia on the immune response in an

experimental model of diabetes in BALB/cByJ and C57Bl/6J mice:

participation of oxidative stress

M. R. Rubinstein, A. M. Genaro and

M. R. WaldCentro de Estudios Farmacológicos y Botánicos

(CEFYBO), Consejo Nacional de Investigaciones

Científicas y Técnicas (CONICET), Primera

Cátedra de Farmacología, Facultad de Medicina,

Universidad de Buenos Aires (UBA), Buenos

Aires, Argentina

Summary

Diabetes is associated with an increased risk of death from infectious disease.

Hyperglycaemia has been identified as the main factor contributing to the

development of diseases associated with diabetes mellitus. However, experi-

mental evidence indicates individual susceptibility to develop complications

of diabetes. In this context, the aim of this work was to study the immune

response in a streptozotocin-induced type 1 diabetes in two mouse strains:

BALB/cByJ and C57Bl/6J. The participation of hyperglycaemia and oxidative

stress was also analysed. Diabetic BALB/cByJ mice showed a decrease in boththe in-vivo and in-vitro immune responses, whereas diabetic C57Bl/6J mice

had higher blood glucose but exhibited no impairment of the immune

response. The influence of hyperglycaemia over the immune response was

evaluated by preincubation of lymphocytes from normal mice in a high

glucose-containing medium. T and B cells from BALB/cByJ mice showed a

decrease in cell viability and mitogen-stimulated proliferation and an

increase in apoptosis induction. An increase in oxidative stress was impli-

cated in this deleterious effect. These parameters were not affected in the T

and B lymphocytes from C57Bl/6J mice. In conclusion, BALB/cByJ mice were

sensitive to the deleterious effect of hyperglycaemia, while C57BL/6J were

resistant. Although an extrapolation of these results to clinical conditions

must be handled with caution, these results highlight the need to contem-plate the genetic background to establish models to study the deleterious

effect of diabetes in order to understand phenotypical variations that are of

clinical importance in the treatment of patients.

Keywords: BALB/cByJ and C57Bl/6J mice, diabetes, high glucose, immune

response, oxidative stress

Accepted for publication 22 October 2012

Correspondence: M. R. Wald, Paraguay 2155

piso 15, CABA, Buenos Aires, Argentina, CP:

1121ABG.

E-mail: [email protected]

Introduction

Diabetes is widely believed to predispose to serious infec-

tions. Evidence from clinical studies supports a causal rela-

tion between diabetes and infections [1–3]. Individuals with

diabetes might be at a higher risk of moderate or severe

infection-related morbidity caused by altered defence

mechanisms [4–7]. However, while some researchers have

shown that infections are more prevalent and have a higher

case fatality rate among patients with diabetes than among

the general population [8,9], others have not observed such

an association [10]. Despite the clinical importance of this

problem, the mechanisms linking diabetes and immuno-

suppression are not well defined.

Hyperglycaemia has been identified as the main factor

contributing to the development of diseases associated with

diabetes mellitus [11–13]. Concerning the risk of infection,

there are substantial clinical data supporting the hypothesis

that hyperglycaemia influences the susceptibility to, and the

outcome of, infectious disease hospitalization [14–17].

Various pathophysiological and biochemical mechanisms

have been proposed to explain the adverse effects of hyper-

glycaemia. Basic and clinical studies have provided new

insights into the role of oxidative stress, suggesting it as a

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Clinical and Experimental Immunology OR I GI NA L A R T I CLE doi:10.1111/cei.12020

319© 2012 British Society for Immunology, Clinical and Experimental Immunology , 171: 319–329



key factor in the pathogenesis of diabetes complications

[18,19]. Evidence indicates that hyperglycaemia results in

an excess of reactive oxygen species (ROS) production, cre-

ating a state of oxidative stress [20–22].

In a previous report, we have analysed the immune

response in an animal model of diabetes and the direct

effect of hyperglycaemia on T and B lymphocyte reactivity.We found that diabetes induces an early decrease in

immunoglobulin (Ig)G levels in the secondary immune

response [23]. Preincubation of lymph node and spleen

cells in a high glucose-containing medium leads to a sig-

nificant time- and dose-dependent decrease in T and B cell

proliferation associated with an increase in oxidative stress

[23].

Inbred mice have been used widely as human disease

models. BALB/cByJ (BALB/c) and C57Bl/6J (C57) inbred

mice are useful models to study the effects of genetic differ-

ences in the pathogenesis of many diseases. These two

mouse strains have differences in both innate [24] and

acquired immunity [25], and have been used extensively to

investigate the immunopathogenesis of several intracellular

infections [26–28]. It has also been reported that both

strains show different metabolic responses to a high-fat diet

[29]. Concerning diabetes, the two strains show inherited

differences in the susceptibility to the effects of streptozo-

tocin in the development of hyperglycaemia: C57 are more

susceptible than BALB/c mice [30].

In this context, the aim of this work was to study the

immune response in diabetic mice of the BALB/c and C57

strains. In particular, we analysed the effect of hyperglycae-

mia and oxidative stress in lymphocyte reactivity. For this

purpose, we used the multiple low doses of streptozotocin(MLD-STZ) animal model. Both mouse strains display

chronic stable hyperglycaemia without a requirement for

insulin therapy and have a lifespan similar to that of normal

mice.

Materials and methods

Animals

Inbred female BALB/cByJ and C57Bl/6J mice were pur-

chased from Facultad de Veterinaria, Universidad de Buenos

Aires, Argentina. Sixty-day-old mice weighing between 20

and 25 g at the beginning of the experiments were used.

Mice were maintained on a 12-h light/dark cycle in a room

with controlled temperature (18–22°C). Animals of each

strain were divided randomly into two experimental groups

of 54 animals each. Additional groups of 25 mice of each

strain were used to analyse the in-vitro effect of hypergly-

caemia. Animals were handled in accordance with the prin-

ciples and guidelines of the Guide for the Care and Use of

Laboratory Animals, US National Research (National Insti-

tutes of Health). Experimental protocols were approved by

the Institutional Committee for use and care of Laboratory

Animal (CICUAL, Facultad de Medicina, Universidad de

Buenos Aires, Argentina).

Experimental diabetes

To induce the diabetic state, one daily dose of streptozo-

tocin (STZ, 40 mg/kg; Sigma Aldrich Co., St Louis, MO,USA) was administered intraperitoneally to the mice for 5

consecutive days [23,31]. Blood glucose measurements were

performed with a One Touch Ultra test strip glucometer

(Lifescan; Johnson-Johnson Co., Milpitas, CA, USA; useful

range, 0·02–0·6 g%) in blood samples obtained from mice

through a small skin incision at the tip of the tail. Glycae-

mia was determined after 2 h of fasting. Mice injected with

the vehicle at the same time were used as the respective

controls.

Immunizations

Sheep red blood cells (SRBC) were used as immunogens to

evaluate the T cell-dependent humoral response and

lipopolysaccharide (LPS) (Sigma Aldrich Co.) was used to

determine the T cell-independent humoral response. For

the SRBC response, mice were immunized intraperitoneally

on day 0 and boosted on day 11 with 0·2 ml of 4% SBRC in

saline. Blood samples were collected for antibody determi-

nation on day 10 (primary response) and day 18 (secondary

response). For LPS, each mouse received an intraperito-

neally injection of 10 mg LPS and blood samples were col-

lected on day 10. Mice injected with the vehicles were used

as controls.

Antibody titres

Quantitative enzyme-linked immunosorbent assay (ELISA)

was performed to determine SRBC- and LPS-specific anti-

bodies, as described previously [32]. Briefly, 96-well plates

(MaxiSorp immunoplates; Nunc, Thermo Fisher Scientific,

Langenselbold, Germany) were coated overnight with SRBC

membranes (7·5 mg) or LPS (1 mg). Dilutions of sera were

added and incubated for 2 h at room temperature and then

with a goat immunoglobulin (Ig)G anti-mouse IgM or IgG,

phosphatase alkaline-conjugated (Sigma Aldrich Co.) and

p-nitrophenylphosphate (Sigma Aldrich Co.) as substrate to

develop coloration that was read at 405 nm. Reactions were

considered positive when optical density values were above

the mean value plus 2 standard deviations (s.d.) of normal

sera (sera from non-immunized vehicle-injected mice).

Cell suspensions and culture conditions

Lymphoid cell suspensions from lymph nodes (axil-

lary, inguinal and mesenteric) or spleen were obtained,

as described previously [23]. The cell culture medium

used was RPMI-1640 (Invitrogen, Carlsbad, CA, USA),

M. R. Rubinstein et al .

320 © 2012 British Society for Immunology, Clinical and Experimental Immunology , 171: 319–329



containing 0·2 g% glucose (Sigma Aldrich Co.). Briefly,

lymphoid organs were removed aseptically and disrupted

through a 1-mm metal mesh and the resulting cell suspen-

sions were filtered through a 10-mm nylon mesh. After

three washes in RPMI-1640 medium, cells were resus-

pended in RPMI-1640 supplemented with 10% of batch-

tested non-stimulatory fetal bovine serum (Invitrogen),2 mM glutamine (Invitrogen), 100 U/ml penicillin (Invit-

rogen) and 100 mg/ml streptomycin (Invitrogen). To

analyse the effect of high concentrations of glucose,

glucose or mannitol or anti-oxidant was added directly to

the medium in the cell culture to yield the final concentra-

tions indicated in each experiment.

Assessment of cell viability

Cell viability was estimated according to the trypan blue

(Sigma Aldrich Co.) exclusion criteria. Normally, freshly

obtained cell viability was higher than 95%.

Proliferation assay

Cells (1·5 ¥ 106 living cells/ml) were settled at a final volume

of 0·2 ml in 96-well flat-bottomed microtitre plates (Nunc)

in triplicate aliquots and incubated at 37°C in a 5% CO2

atmosphere. The T cell-selective mitogen concanavalin A

(Con A; Sigma Aldrich Co.) and the mitogen selective for B

cells, lipopolysaccharide (LPS) were used [23]. Proliferation

was measured by adding 0·75 mCi [3H]-thymidine (specific

activity: 20 Ci/mmol; Perkin-Elmer, Boston, MA, USA) for

the last 18h of culture. Thymidine incorporation was meas-ured by scintillation counting. The mean of triplicate deter-

minations was calculated for each lectin concentration.

Detection of lymphocyte apoptosis

Apoptosis was estimated by labelling DNA and analysing

chromatin morphology by measuring subdiploid DNA

content by flow cytometric analysis, according to a modifi-

cation of the Nicoletti method [33], as described previously

[23].

Lymph node and spleen cells were harvested after 24 h

incubation, washed and fixed overnight with cold 70%

ethanol. The cells were then stained in the dark for 30 min

at room temperature with propidium iodide (PI) (Sigma

Chemical Co.) and analysed by flow cytometry using a fluo-

rescence activated cell sorter (FACS)can flow cytometer

(Becton Dickinson, Franklin Lakes, NJ, USA) with log

amplification and FACScan research software. For each

sample, 10 000 cells were acquired and analysed using the

WinMDI version 2·9 program. Apoptotic nuclei appeared as

a broad hypodiploid DNA peak which was easily discrimi-

nated from the narrow peak of normal (diploid) DNA

content [23].

Measurement of intracellular reactive oxygen species

(ROS) generation

ROS produced within lymphocytes were detected with the

membrane-permeant probe 2′,7′-dichlorofluorescein diac-

etate (DCFH-DA; Sigma Aldrich Co.) [34]. The lym-

phocytes (1 ¥ 106

living cells) were incubated in the dark with 5 mM DCFH-DA for 15 min at 37°C. The intensity of

dichlorofluorescein (DCF) fluorescence in the supernatant

was measured with an excitation wavelength of 485 nm, and

an emission wavelength of 530 nm (Hitachi F2000 Fluores-

cence Spectrophotometer; Hitachi, Tokyo, Japan). Intracel-

lular ROS production was calculated from a DCF standard

curve (1–40 nmol/ml).

Colorimetric assay for lipid peroxidation

Malondialdehyde (MDA), a marker for lipid peroxidation

formed by the breakdown of polyunsaturated fatty acids,was determined [35]. Lymphocytes (2 ¥ 107 living cells)

were resuspended in 5% trichloroacetic acid and incubated

with 15% trichloroacetic acid, 0·375% thiobarbituric acid

(Sigma Aldrich Co.) and 0·25 N hydrochloric acid. The

samples were heated for 15 min in a boiling waterbath.

After cooling, the flocculent precipitate was removed by

centrifugation and the absorbance in the supernatant was

determined at 535 nm. A standard plot for MDA was pre-

pared using 1,1,3,3-tetraethoxypropane (Sigma Aldrich

Co.).

Measurement of the intracellular total anti-oxidantcapacity (TAC)

TAC within lymphocytes was determined by using the

ABTS [2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic

acid)] anti-oxidant assay [36]. Briefly, acetate buffer and the

lymphocyte samples (7 ¥ 106 living cells) were mixed in 96-

well plates (MaxiSorp immunoplates; Nunc) with ABTS+

solution 10 mmol/l (0·549 g of ABTS (MP Biomedicals) in

acetate buffer 30 mmol/l pH 3·6 and 2 mmol/l hydrogen

peroxide). Samples were read at 660 nm on a plate reader.

Results were calculated using a reference curve based on the

anti-oxidant Trolox (Sigma-Aldrich Co) as a standard.

Measurement of glutathione

Glutathione (GSH) analysis was achieved using the GSH

reductase recycling assay as described by Tietze, modified by

Baker [37,38]. Lymphocytes (10 ¥ 107 living cells) were

resuspended in 0·5% trichloroacetic acid, and for quantifi-

cation of oxidized glutathione (GSSG) the samples were

incubated for 1 h with 1 M 3-vinil pyridine, a scavenger

of reduced glutathione (GSH). Then, the reaction mixture

[1 mM 3,3′-dithio-bis(6- nitrobenzoic acid (DTNB)],

Hyperglycaemia and immune response




200 U/ml glutathione reductase (GR), 1 mM nicotinamide

adenine dinucleotide phosphate (NADPH) and 100 mM

sodium phosphate buffer pH 7·5 with 1 mM ethylemedi-

amine tetraacetic acid (EDTA)] was added. Absorbance was

recorded at 2-min intervals for 10 min at 405 nm in an

ELISA plate reader (Bio-Rad, Hercules, CA, USA). Total and

GSSG were determined in each sample in a 96-well microti-tre plate in duplicate. The GSH was calculated as the differ-

ence between the total glutathione and GSSG.

Statistical analysis

Group differences were tested by one- or two-way analyses

of variance (anova), with treatment and time or treatment

and strain as main factors. When the interaction was signifi-

cant, simple effects analysis was carried out. Post-hoc com-

parisons were applied to compare the data between the

groups with the Student–Newman–Keuls (SNK) test. For

antibody titres, the Kruskal–Wallis test was performed fol-

lowing Conover non-parametric post-hoc tests. All tests

were two-sided, and a P -value of 0·05 was considered statis-

tically significant. Statistical and data analyses were per-

formed using GraphPad Prism version 5·00 for Windows

(GraphPad Software, San Diego, CA, USA) and Infostat

Software (Cordoba University, Argentina).

Results

Glucose determination

Hyperglycaemia has been identified as the main factor con-

tributing, through either direct or indirect mechanisms, todiabetic pathogenesis by producing biochemical and meta-

bolic alterations that lead to both functional and structural

alterations. Therefore, we determined glycaemia in both

BALB/c and C57 mice until 6 months of diabetes induction.

As shown in Fig. 1, diabetic mice displayed an increase in

blood glucose concentrations, but C57 showed higher levels

of glucose than BALB/c mice (0·16–0·3 g% versus 0·15–

0·2 g%) (RM anova; main effects: strain F (3,44) = 48·25,

P < 0·0001). However, survival without insulin administra-

tion was similar in both strains.

Antibody production after in-vivo immunization

To investigate if changes in the humoral response are

present in diabetic mice, we examined the antibody produc-

tion after immunization with SRBC (a T cell-dependent

antigen) and LPS (a T cell-independent antigen) at early (15

days and 1 month) and late (6 months) periods of diabetes

induction. To analyse the primary immune response, spe-

cific antibody titres of the IgM type were evaluated. In

BALB/c mice, IgM production after LPS and SRBC immu-

nization was not significantly different between control and

diabetic animals after 15 days and 1 month of diabetes

induction, whereas a significant decrease was observed after

6 months of diabetes induction (Fig. 2a,b). However, during

the secondary response, titres of anti-SRBC IgG were lower

in animals with diabetes than in controls after 1 month of

diabetes induction (Fig. 2c). In contrast, diabetic C57 mice

displayed a decrease in the production of anti-SRBC IgGonly after 6 months of diabetes induction (Fig. 2a–c)

(Kruskal–Wallis test IgM LPS: BALB/c P = 0·0045; C57 P

not significant; IgM GRc: BALB/c P = 0·0093; C57 P not sig-

nificant; IgG GRc: BALB/c P = 0·0084; C57 P = 0·0273).

Lymphocyte reactivity

To investigate if in-vivo antibody production is correlated

with in-vitro lymphocyte reactivity, we evaluated the

mitogen-induced T and B cell proliferation. Due to the

well-known lymphoid profile, cell suspension from lymph

nodes was used for the stimulation of the T selective

mitogen ConA, while spleen lymphocyte suspensions were

used to evaluate LPS (B cell selective) mitogen effect. The

maximal proliferation was reached at 1 mg/ml of ConA and

25 mg/ml of LPS for both control and diabetic mice at all

times tested (data not shown). The maximal mitogen-

induced T and B lymphocyte proliferation was altered in

BALB/c diabetic animals 6 months after diabetes induction

(Fig. 3) (simple effect analysis, 6 months, T proliferative

response: P < 0·0001; B proliferative response: P < 0·0001).

However, we found no differences for B and T lymphocyte

reactivity in C57 mice at any of the times tested.

0 50 100 150 200

0·01

0·10

0·15

0·20

0·25

0·30

BALB/c diab C57 diab

**

**** **

** ** **

**** **

**

BALB/c control C57 control

****

** **

**

** ****

** **

**

**

**

##

##

#### ##

#### ##

## ## ##

Days after treatment

G l y c a

e m i a ( g % )

Fig. 1. Glycaemia values in BALB/c and C57 normal and diabetic

mice. Average glycaemia values versus days after streptozotocin

treatment in BALB/c and C57 mice. Data shown are themean standard error of the mean of 12 animals in each group.

Statistical significance was determined with two-way repeated-

measures analysis of variance with a 4 ¥ 12 design followed by

Student–Newman–Keuls (SNK) post-test. **P < 0·01 with respect to

control mice; ##P < 0·01 with respect to BALB/c diabetic mice.





Effect of high glucose on in-vitro lymphocyte

proliferation

In order to gain insight into the potential mechanisms

involved in this alteration, we then evaluated the influence

of hyperglycaemia on the immune response. Lymph node

and spleen lymphocytes isolated from normal BALB/c and

C57 mice were exposed to media containing increasing con-

centrations of glucose for 24 h. After these treatments,

viability, apoptosis and mitogen-induced proliferative

response were determined. As seen in Fig. 4, after 24 h of

culture with high glucose concentrations, T and B lym-

phocytes from BALB/c mice showed a significant decreasein mitogen-stimulated proliferation (Fig. 4a,b, simple effect

analysis, T and B proliferative response: P < 0·0001 for each

one) and cell viability (Fig. 4c,d, simple effect analysis, T

and B proliferative response: P < 0·0001 for each one) and

an increase in apoptosis induction (Fig. 4e, T lymphocytes:

P = 0·0061; B lymphocytes: P = 0·0028). Interestingly, these

parameters were not affected in T and B lymphocytes from

normal C57 mice exposed to 0·5 and 1 g% of glucose;

however, a significant decrease was found with 2 g% of

glucose (Fig. 4). Unlike d-glucose, 0·5 and 1 g% of mannitol

did not change T and B cell proliferation, viability or apop-

tosis in BALB/c lymphocytes when compared with controls,but with 2 g% of mannitol a significant alteration of these

parameters was observed (Table in Fig. 4). Similar results

were observed in C57 mice. These findings suggest that the

effect of 0·5 and 1 g% of glucose, but not of 2 g%, is not the

result of high osmolarity. Thus, the following studies were

performed with 0·5 and 1 g% of glucose.

Oxidative stress participation in hyperglycaemia effects

Oxidative stress in lymph node and spleen lymphocytes was

analysed by the detection of ROS and by the measurement

of lipid peroxidation performed by MDA production. After

24 h incubation with high glucose, T and B lymphocytes

from BALB/c but not from C57 mice showed an increase in

ROS production (simple effect analysis, T lymphocytes and

B lymphocytes: BALB/c P < 0·0001; C57 P not significant

for each one) and lipid peroxidation (MDA production:

simple effect analysis, T lymphocytes: BALB/c P = 0·0026;

C57 P not significant; B lymphocytes: BALB/c P = 0·0002;

C57 P not significant) in comparison with control cells

(Fig. 5). When the same concentration of mannitol was

used, no enhancement of oxidative stress was observed

(data not shown).

Anti-LPS IgM

15 Days 1 Month 6 Months

0

1

2

3

*

- l o g t i t r e

Anti-SRBC IgM


0

1

2

3

*

- l o g t i t r e

Anti-SRBC IgG


0

1

2

3

4

*

*

*

- l o g t i t r e

BALB/c control BALB/c diab

C57 control C57 diab

(a)

(b)

(c)

Fig. 2. Antibody production following sheep red blood cells (SRBC)

and lipopolysaccharide (LPS) immunization in BALB/c and C57

control and diabetic mice. Antibody titres in controls and at 15 days, 1

month and 6 months after diabetes induction in BALB/c and C57

mice. (a) Anti-LPS immunoglobulin (Ig)M production. (b)

Anti-SRBC IgM production. (c) Anti-SRBC IgG production. Data

shown are the mean standard error of the mean of three animals in

each group. Statistical significance was determined with theKruskal–Wallis test followed by Conover post-test. *P < 0·05 with

respect to control mice.





To corroborate if the decrease in T and B cell reactivity of

lymphocytes from BALB/c mice in the presence of high

glucose was due to an increase in oxidative stress, we incu-bated the cells in the presence of N-acetylcysteine (NAC)

(5 mM). Figure 6 shows that the presence of NAC prevented

the effect of high glucose on proliferation, viability

and apoptosis in both T and B lymphocytes from

BALB/c mice (proliferation, T and B lymphocytes, main

effects: glucose ¥ anti-oxidant interaction P = 0·0009 andP < 0·0001, respectively; viability, T and B lymphocytes,

main effects: glucose ¥ anti-oxidant interaction P = 0·0139

and P < 0·0001, respectively; apoptosis: T and B

T lymphocytes

15 Days 1 Month 3 Months 6 Months0

50 000

100 000

150 000

*

P r o l i f e r a t i o n ( d p m )

B lymphocytes

15 Days 1 Month 3 Months 6 Months0

10 000

20 000

30 000

40 000

*

P

r o l i f e r a t i o n ( d p m )

BALB/c control BALB/c diab C57 control C57 diab

(a) (b)

Fig. 3. Mitogen-induced proliferative response in T and B cells from controls and at 15 days, 1, 3 and 6 months of diabetes in BALB/c and C57

mice. (a) T lymphocytes stimulated with 1 mg/ml of concanavalin A. (b) B lymphocytes stimulated with 25 mg/ml of lipopolysaccharide (LPS).

Results shown are the mean standard error of the mean of three independent experiments performed in triplicate. Statistical significance was

determined with two-way analysis of variance with a 4 ¥ 4 design followed by Student–Newman–Keuls (SNK) post-test. *P < 0·05 with respect to

cells from control mice.

T lymphocytes

Control 0·5 1 2

50 000

0

100 000

150 000

200 000

(a) (b)

(d)(c)

(e)

**

**

*

**

Glucose (g%)


B lymphocytes

Control 0·5 1 20

20 000

40 000

60 000

**

**

**

Glucose (g%)


T lymphocytes

Control 0·5 1 20

20

40

60

80

100

** **

**

**

Glucose (g%)

V i a b i l i t y

( % )

B lymphocytes

Control 0·5 1 20

20

40

60

80

100

** ****

Glucose (g%)

V i a b i l i t y

( % )

T lymphocytes B lymphocytes0

10

20

30

40

control

glucose 1 g% glucose 2 g%

****

*

**

A p o p t o s i s ( % )

BALB/c C57

Cultured media addedMannitol

0·5 g %

Mannitol

1 g %

Mannitol

2 g %

Tlymphocytes 111 ± 6 ·7 97 ± 4 ·6 72 ± 9 ·2 *Proliferation

(% ofcontrol)

Blymphocytes 101 ± 7 95 ± 8·5 74 ± 4·4 *

75 ± 9 *

Lymph nodelymphocytes 105 ± 10 96 ± 5

9·9 ± 4·1

13·7 ± 3·22

98 ± 699 ± 9

8·9 ± 2·4

13·1 ± 3·5

76 ± 8 *Viability(% of

control) spleenlymphocytes

Lymph nodelymphocytes 19·2 ± 5·7

18·9 ± 4·1

Apoptosis

(%) spleenlymphocytes

Fig. 4. Effect of high concentrations of glucose

and mannitol on proliferation, viability and

apoptosis of T and B normal lymphocytes. T

lymphocytes (a,c) or B lymphocytes (b,d) from

BALB/c and C57 mice were incubated for 24 h

in RPMI-1640 either with or without (control)

addition of glucose in the culture medium.

Proliferation (a,b) and cell viability (c,d) were

determined. (e) BALB/c lymphocyte apoptosis.

Table: effect of high concentrations of mannitol

on T and B cells from BALB/c mice. Results

shown are the mean standard error of the

mean of five independent experiments

performed in triplicate for proliferation and

viability and of three independent experiments

performed in triplicate for apoptosis. Statistical

significance was determined with two-way

analysis of variance with a 2 ¥ 4 design,

followed by Student–Newman–Keuls (SNK)

post-test for proliferation and viability and with

one-way analysis of variance for three factors

for apoptosis and for mannitol effect. *P < 0·05

and **P < 0·01 with respect to control values

(standard medium alone).





T lymphocytes

Control 0·5 10

2

4

6

8

10

**

(a) (b)

(c) (d)

**

Glucose (g%)

R O

S p r o d u c t i o n

( n m o l D C F / 1 0 6

c e l l s )

B lymphocytes

Control 0·5 10

2

4

6

8

10

**

**

Glucose (g%)

R O

S p r o d u c t i o n

( n m o l D C F / 1 0 6

c e l l s )

T lymphocytes

Control 0·5 10·0

0·2

0·4

0·6

0·8

**

Glucose (g%)

M D A p r o d u c t i o n

( n

m o l M D A / 1 0 7

c e l l s )

B lymphocytes

Control 0·5 10·0

0·5

1·0

1·5

2·0

2·5

Glucose (g%)

**

M D A p r o d u c t i o n

( n

m o l M D A / 1 0 7

c e l l s )

BALB/c C57

Fig. 5. Effect of high concentrations of glucoseon oxidative stress. Reactive oxygen species

(ROS) production and lipid peroxidation.

Lymph node (a,c) and spleen (b,d) lymphocytes

from BALB/c and C57 mice were incubated for

24 h in RPMI-1640 either with or without

(control) addition of glucose in the culture

medium. ROS (a,b) or malondialdehyde (MDA)

production (as a measure of lipid peroxidation)

(c,d) were determined. Data shown are the

mean standard error of the mean of five

independent experiments performed in

duplicate. Statistical significance was

determined with two-way analysis of variance

with a 2 ¥ 3 design followed by Student–Newman–Keuls (SNK) post-test.

**P < 0·01with respect to control values.

T lymphocytes

Control 0·5 10

50 000

100 000

150 000

(a) (b)

(c) (d)

(e) (f)

** **

**

Glucose (g%)


B lymphocytes

Control 0·5 10

10 000

20 000

30 000

**

Glucose (g%)

P r

o l i f e r a t i o n ( d p m )

T lymphocytes

Control 0·5 1

0

50

100

150

****

Glucose (g%)

V i a b i l i t y ( % )

B lymphocytes

Control 0·5 1

0

20

40

60

80

100

****

Glucose (g%)

V i a b i l i t y ( % )

T lymphocytes

Control 0·5 10

5

10

15

20

25

**

**

Glucose (g%)

A p o p t o s i s ( % )

B lymphocytes

Control 0·5 10

10

20

30

**

**

Glucose (g%)

A p o p t o s i s ( % )

– NAC + NAC

Fig. 6. N-acetylcysteine (NAC) action on the

effect of high glucose on mitogen-induced

proliferative response, viability and apoptosis in

T and B cells from normal BALB/c mice. Lymph

node (a,c,e) and spleen lymphocytes (b,d,f)

from BALB/c mice were incubated for 24 h in

RPMI-1640 either with or without (control)

the addition of glucose or NAC in the culture

medium. Proliferation (a,b), cell viability (c,d)

and apoptosis (e,f) were determined. Results

shown are the mean standard error of the

mean of five independent experiments

performed in triplicate for proliferation and

viability and of three independent experiments

performed in triplicate for apoptosis. Statistical

significance was determined with two-way

analysis of variance with a 2 ¥ 3 design followed

by simple effects analysis. **P < 0·01 with

respect to control values.





both functional and structural alterations [11–13,44]. In

fact, in a previous work, we have observed a negative corre-

lation between plasma glucose levels and T and B cell prolif-

eration [45]. Diabetic BALB/c mice exposed to chronic mild

stress had a sustained increase in blood glucose levels. In

these animals, a decrease in T and B cell proliferation was

observed earlier, after 3 and 6 weeks under stress exposure,respectively [45]. We have also reported previously that

in-vitro high glucose levels can alter the reactivity of both

normal T and B lymphocytes in a time- and concentration-

dependent interaction manner [23]. In addition, we have

observed that the presence of high glucose in lymph node

and spleen lymphocyte cultures decreases cell viability with

a higher percentage of apoptotic cells. We have also found

that an increase in oxidative stress, in particular an excess

of reactive oxygen species (ROS) production, would be

implicated in these deleterious effects [23]. Under normal

conditions, these toxic species are produced by cellular

metabolism and neutralized by endogenous anti-oxidant

defences. However, in adverse conditions, such as hypergly-

caemia, cellular defences might be insufficient, leading to

damage of cellular components [46]. Glutathione (GSH,

L-glutamyl-L-cysteinyl-glycine) is the most abundant non-

protein thiol in mammalian cells and is one of the major

cellular defences against ROS and free radicals [47]. GSH is

essential for cell survival and has been implicated as an

important regulator of T cell function and proliferation

[39]. Furthermore, GSH depletion is necessary for lym-

phocyte apoptosis [47]. N-acetylcysteine (NAC) is a small

thiol compound that leads to increased glutathione levels

[39]. In this study we found that co-incubation with the

anti-oxidant NAC prevented the deleterious effects of highglucose.

Interestingly, diabetic C57 mice showed impaired IgG

production only after 6 months of diabetes induction.

These results indicate that although diabetic C57 mice

have higher plasma glucose levels than BALB/c mice, the

immune response is less affected. In addition, lymphocyte

reactivity was not altered at this time. In accordance with

these findings, proliferation, viability and apoptosis were

not altered by high glucose incubation. Moreover, we did

not find an increase in oxidative stress under this condition.

To assess the involvement of anti-oxidant mechanisms, we

determined the TAC of lymphocytes. In the presence of

high glucose, BALB/c lymphocytes show a decrease in TAC;

however, an increment was observed in those from C57.

The same pattern of response was observed for the

GSH : GSSG ratio. Changes in the intracellular GSH : GSSG

balance are considered major determinants in the redox

status/cell signalling [48]. Taking these results into account,

it can be postulated that the increase in the GSH : GSSG

ratio observed in lymphocytes from C57 mice may consti-

tute a mechanism of protection against the deleterious

effects of oxidative stress induced by high glucose. Consist-

ent with this observation, an up-regulation of anti-oxidant

enzymes was reported as an adaptation to oxidative stress

during the formation of acquired bleomycin resistance in a

cell line [49]. However, the involvement of other anti-

oxidant mechanisms that would protect C57 lymphocytes

cannot be ruled out. Further studies are necessary to eluci-

date the precise biochemical and molecular mechanisms

involved.In conclusion, BALB/cByJ mice are more sensitive to the

deleterious effect of hyperglycaemia on the immune

response than C57Bl/6J mice. Qi et al . [50] characterized

the severity of diabetic nephropathy in six inbred mouse

strains, including C57Bl/6J, DBA/2J, FVB/NJ, MRL/MpJ, A/J

and KK/HlJ mice, by inducing diabetes by multiple low-

dose streptozotocin injection and found that KK/HlJ mice

seemed to be relatively resistant to STZ-induced hypergly-

caemia with lower levels of blood glucose. Without insulin

supplementation, most diabetic mice survived for more

than 25 weeks. After that, the mortality increased in most

strains studied, especially in DBA/2J, KK/HlJ and A/J mice.

In contrast, C57Bl/6J mice seemed to tolerate persistent

hyperglycaemia well, with a group of diabetic C57Bl/6J

mice surviving for longer than 45 weeks despite fasting

glucose levels of 0·3–0·6 g%. With respect to renal injury,

DBA/2J and KK/HlJ mice developed significantly more

albuminuria than C57Bl/6J, MRL/MpJ and A/J mice. These

results highlight the need to contemplate the genetic back-

ground in order to establish models to study the deleterious

effect of diabetes. These results also open the possibility of

using mouse models to map genes for susceptibility to dif-

ferent diabetic complications. Nowadays, individual vari-

ability is an important challenge in current clinical practice

and phenotypical variations are of clinical importance inthe treatment of patients. Knowledge of the genetic deter-

minants of the disease pathogenesis is not always available.

Although the extrapolation of these results to clinical con-

ditions should be handled with caution, it is important to

emphasize the importance of conducting studies with large

samples that can provide better estimates of the magnitude

of the genetic contributions to major individual variability,

and deliver solid results to decision-making in clinical prac-

tice, both preventive and therapeutic.

Acknowledgements

This study was supported by grants from CONICET, PIP

11220080100281. The authors wish to thank Daniel

Gonzalez and Marcela Marquez for their valuable help in

the animal care, María Rosa Gonzalez Murano for technical

support and Patricia Fernandez for secretarial assistance.

We thank LifeScan Argentina for supplying One Touch

Ultra strips for the glucometer.

Disclosure

The authors declare that there are no conflicts of interest.





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